Controlled 3D culture of human periosteum derived cells for Tissue Engineering

The field of tissue engineering aims to provide solutions for the regeneration of organs and tissues. It has long been recognised that biomimetic approaches can provide viable solutions to tackle the current challenges in the field. The use of perfusion bioreactors has shown great promise for the development of three dimensional (3D) tissue engineered constructs especially when scaling them up. Indeed, bioreactors provide enhanced nutrient delivery to the cells and extracellular matrix deposition during culture. This is important since it promotes cell survival in engineered constructs, becoming crucial In the case of large scale constructs were diffusion limitation become detrimental. Many studies employed perfusion bioreactors, however, most of them were carried out in non-controlled set ups, hence hindering the robustness and reproducibility of the bioprocess. In this study, we present the results of a series of studies performed in a controlled perfusion bioreactor setup. Titanium scaffolds were seeded with hPDCs (200000 cells/60µL) and cultured under a 0.1 mL/min perfusion flowrate for 3 weeks resulting in a 3D tissue construct of around 1.6 million cells. Sampling of the growth medium was regularly carried out in order to measure metabolite consumption and waste production. By measuring lactate and glucose concentration during culture, the cells’ production or consumption of these metabolites was monitored and plotted over time. At the end of the culture, Live/Dead staining of the constructs (cells + scaffold) was performed. The hPDCs grown using this method showed metabolic phenotypes of proliferating cells. The Live/Dead staining showed bridging of the cells across the pores of the scaffolds, showing tissue growth and 3D extra cellular matrix deposition. These results have proven to be reproducible throughout multiple repetitions of the experiments. This study goes one step further in developing robust, reproducible 3D perfusion bioprocesses.status: publishe